RESUMEN
The cell-free method is suitable for rapid and economical production of therapeutic proteins, since it is an open system, which allows us to control the reaction microenvironment to promote folding, solubility of proteins and maximize the protein yield. Consensus interferon is a newly developed type I interferon, a rapid-acting version of interferon that appears more potent than the currently approved pegylated version. Our work aimed to synthesize human consensus interferon-alpha (cIFN-α) in cell-free protein expression system of Escherichia coli cells origin. The cloned cIFN-α gene in pET101/D-TOPO expression system was used in cell-free IFN production. The system was tested by using a standard construct, GFP (green fluorescent protein) gene was cloned into pIVEX2.3 vector; this gene and our gene, both are under the T7 promoter transcriptional control. The synthesis of active cIFN-α gradually increased from 2 to 6 h of the reaction, also reducing the temperature of incubation to ≤ 30°C maximized its solubility. After purification on nickel-nitrilotriacetate acid (Ni-NTA) resin, the yield of cIFN-α was 400 µg/ml cell-free reaction solution. The resultant cIFN-α was fully biologically active as demonstrated by its anti-cancer effect and immunoassay signals.
Asunto(s)
Antineoplásicos/farmacología , Escherichia coli/genética , Interferón-alfa/genética , Interferón-alfa/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Antineoplásicos/aislamiento & purificación , Antineoplásicos/metabolismo , Escherichia coli/citología , Humanos , Interferón-alfa/aislamiento & purificación , Neoplasias/tratamiento farmacológico , Plásmidos/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Hepatitis C virus is a major public health problem leading to cirrhosis and increased risk for development of hepatocellular carcinoma, both leading indications for liver transplantation. Egypt has the highest prevalence of hepatitis C of worldwide. Anti-HCV antibody is usually detected by enzyme immunoassay (EIA). Microarray analysis of 8 Anti-HCV antibody isotypes and 5 HCV peptides considered separately on 5 different matrixes was carried out on 50 hepatitis C Egyptian patients. The optimal substrate kind was chosen based on the greatest amount of antibody bonded with the minimal background. Mercaptosilane activated slides, agarose-slides and polyvinylidene difluoride membranes were the best substrates, while polyacrylamide and nitrocellulose membrane were less sensitive. IgM isotype gave the weakest signals in all patients whatever the substrate type used for antibody immobilization while IgG (total) and its subtypes IgG2, IgG3 and IgG(4) gave the strongest signals with most of the substrates follows by IgA(1), IgG(1) and total IgA, respectively. The results demonstrate that IgG2, IgG3 and IgG(4) are the dominant IgG subtypes, which may indicate that HCV patient immune response shift toward Th-2 immunity. The microarrays permitted the simultaneous serodetection of hepatitis C virus core and envelope peptides by using corresponding rabbit anti-peptides. Hepatitis C virus core and envelope peptides 1, 2, 3, 4 and 5 showed strong signals on all the used substrates except for polyacrylamide slides and nitrocellulose membranes.
Asunto(s)
Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/sangre , Hepatitis C Crónica/inmunología , Isotipos de Inmunoglobulinas/sangre , Análisis por Matrices de Proteínas/métodos , Egipto/epidemiología , Ensayo de Inmunoadsorción Enzimática , Femenino , Hepatitis C Crónica/sangre , Hepatitis C Crónica/epidemiología , Humanos , MasculinoRESUMEN
The ability of different local isolates in addition to some isolates from Germany to degrade kerosene in liquid medium was studied. The results showed that the percent of kerosene degradation varied among the different organisms and that 59-94% of kerosene was degraded after 21d. Two local isolates (Pseudomonas sp. AP and Pseudomonas sp. CK) and one German isolate (Gordonia sp. DM) were selected for this study. The addition of wheat bran, as co-substrate, stimulated the kerosene degradation by the two local strains, while glucose inhibited the degradation rate using the three organisms with different rates. Ammonium nitrate and urea was the best nitrogen sources. The use of superphosphate (as phosphorus source) in the presence of urea stimulates the degradation rate. It was also observed that the addition of 1% surfactants, like Triton X-100, Igepal, Tergitol, or Tween 20 and 80 enhanced the kerosene degradation. The degradation percent lied between 94% and 98%. The ability of the tested organisms to degrade kerosene concentration from 2% to 8% was evaluated. It was found that the three organisms degraded about 65-85% from 8% kerosene after 21d. The use of rice straw-immobilized cells reduced the time of degradation and enhanced the degradation ability of the organisms. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed the presence of a common protein band when the tested organisms were grown on kerosene.